Promoter sequences providing male tissue-preferred expression in plants

ABSTRACT

Promoter sequences and their essential regions are identified which provide for male tissue-preferred expression. The nucleotide sequences are useful in mediating male fertility in plants.

This application is a continuation of previously filed and co-pendingapplication U.S. Ser. No. 10/058,566, filed Jan. 28, 2002, which claimsbenefit under 35 U.S.C.§119(e) of previously filed and co-pendingprovisional application 60/267,527, filed Feb. 8, 2001.

BACKGROUND OF THE INVENTION

Development of hybrid plant breeding has made possible considerableadvances in quality and quantity of crops produced. Increased yield andcombination of desirable characteristics, such as resistance to diseaseand insects, heat and drought tolerance, along with variations in plantcomposition are all possible because of hybridization procedures. Theseprocedures frequently rely heavily on providing for a male parentcontributing pollen to a female parent to produce the resulting hybrid.

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinating if pollenfrom one flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

In Brassica, the plant is normally self sterile and can only becross-pollinated. In self-pollinating species, such as soybeans andcotton, the male and female plants are anatomically juxtaposed. Duringnatural pollination, the male reproductive organs of a given flowerpollinate the female reproductive organs of the same flower.

Maize plants (Zea mays L.) present a unique situation in that they canbe bred by both self-pollination and cross-pollination techniques. Maizehas male flowers, located on the tassel, and female flowers, located onthe ear, on the same plant. It can self or cross pollinate. Naturalpollination occurs in maize when wind blows pollen from the tassels tothe silks that protrude from the tops of the incipient ears.

A reliable method of controlling fertility in plants would offer theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system and where a female sterility system would reduceproduction costs.

The development of maize hybrids requires the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Pedigree breeding and recurrent selection are two of thebreeding methods used to develop inbred lines from populations. Breedingprograms combine desirable traits from two or more inbred lines orvarious broad-based sources into breeding pools from which new inbredlines are developed by selfing and selection of desired phenotypes. Ahybrid maize variety is the cross of two such inbred lines, each ofwhich may have one or more desirable characteristics lacked by the otheror which complement the other. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which have commercial potential. The hybrid progeny of thefirst generation is designated F₁. In the development of hybrids onlythe F₁ hybrid plants are sought. The F₁ hybrid is more vigorous than itsinbred parents. This hybrid vigor, or heterosis, can be manifested inmany ways, including increased vegetative growth and increased yield.

Hybrid maize seed can be produced by a male sterility systemincorporating manual detasseling. To produce hybrid seed, the maletassel is removed from the growing female inbred parent, which has beenplanted in alternating rows with the male inbred parent. Consequently,providing that there is sufficient isolation from sources of foreignmaize pollen, the ears of the female inbred will be fertilized only withpollen from the male inbred. The resulting seed is therefore hybrid andwill form hybrid plants.

Environmental variation in plant development can result in plantstasseling after manual detasseling of the female parent is completed.Or, a detasseler might not completely remove the tassel of a femaleinbred plant. In any event, the result is that the female plant willsuccessfully shed pollen and some female plants will be self-pollinated.This will result in seed of the female inbred being harvested along withthe hybrid seed, which is normally produced. Female inbred seed is notas productive as F1 seed. In addition, the presence of female inbredseed can represent a germplasm security risk for the company producingthe hybrid.

Alternatively, the female inbred can be mechanically detasseled bymachine. Mechanical detasseling is approximately as reliable as handdetasseling, but is faster and less costly. However, most detasselingmachines produce more damage to the plants than hand detasseling. Thus,no form of detasseling is presently entirely satisfactory, and a needcontinues to exist for alternatives, which further reduce productioncosts and eliminate self-pollination in the production of hybrid seed.

A reliable system of genetic male sterility would provide advantages.The laborious detasseling process can be avoided in some genotypes byusing cytoplasmic male-sterile (CMS) inbreds. In the absence of afertility restorer gene, plants of a CMS inbred are male sterile as aresult of factors resulting from the cytoplasmic, as opposed to thenuclear, genome. Thus, this characteristic is inherited exclusivelythrough the female parent in maize plants, since only the femaleprovides cytoplasm to the fertilized seed. CMS plants are fertilizedwith pollen from another inbred that is not male-sterile. Pollen fromthe second inbred may or may not contribute genes that make the hybridplants male-fertile. Usually seed from detasseled normal maize and CMSproduced seed of the same hybrid must be blended to insure that adequatepollen loads are available for fertilization when the hybrid plants aregrown and to insure diversity.

There can be other drawbacks to CMS. One is an historically observedassociation of a specific variant of CMS with susceptibility to certaincrop diseases. This problem has discouraged widespread use of that CMSvariant in producing hybrid maize and has had a negative impact on theuse of CMS in maize in general.

One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465and 4,727,219 to Brar, et al. However, this form of genetic malesterility requires maintenance of multiple mutant genes at separatelocations within the genome and requires a complex marker system totrack the genes and make use of the system convenient. Patterson alsodescribed a genic system of chromosomal translocations which can beeffective, but which are complicated. U.S. Pat. Nos. 3,861,709 and3,710,511.

Many other attempts have been made to improve on these drawbacks. Forexample, Fabijanski, et al., developed several methods of causing malesterility in plants (see EPO 89/3010153.8 publication no. 329,308 andPCT application PCT/CA90/00037 published as WO 90/08828). One methodincludes delivering into the plant a gene encoding a cytotoxic substanceassociated with a male tissue specific promoter. Another involves anantisense system in which a gene critical to fertility is identified andan antisense to the gene inserted in the plant. Mariani, et al. alsoshows several cytotoxic and antisense systems. See EP 89/401, 194. Stillother systems use “repressor” genes which inhibit the expression ofanother gene critical to male sterility. PCT/GB90/00102, published as WO90/08829.

A still further improvement of this system is one described at U.S. Pat.No. 5,478,369 (incorporated herein by reference) in which a method ofimparting controllable male sterility is achieved by silencing a genenative to the plant that is critical for male fertility and replacingthe native DNA with the gene critical to male fertility linked to aninducible promoter controlling expression of the gene. The plant is thusconstitutively sterile, becoming fertile only when the promoter isinduced and its attached male fertility gene is expressed.

As noted, an essential aspect of much of the work underway with malesterility systems is the identification of genes impacting malefertility.

Such a gene can be used in a variety of systems/to control malefertility including those described herein. Previously, a male fertilitygene has been identified in Arabidopsis thaliana and used to produce amale sterile plant. Aarts, et al., “Transposon Tagging of a MaleSterility Gene in Arabidopsis”, Nature 363:715-717 (Jun. 24, 1993). U.S.Pat. No. 5,478,369 discloses therein one such gene impacting malefertility. In the present invention the inventors provide novel DNAmolecules and the amino acid sequence encoded that are critical to malefertility in plants. The inventors also provide a promoter of the geneand its essential sequences. These can be used in any of the systemswhere control of fertility is useful, including those described above.

Thus, one object of the invention is to provide a nucleic acid sequence,the expression of which is critical to male fertility in plants.

Another object of the invention is to provide a DNA molecule encoding anamino acid sequence, the expression of which is critical to malefertility in plants.

Yet another object of the invention is to provide a promoter of suchnucleotide sequence and its essential sequences.

A further object of the invention is to provide a method of using suchDNA molecules to mediate male fertility in plants.

Further objects of the invention will become apparent in the descriptionand claims that follow.

SUMMARY OF THE INVENTION

This invention relates to nucleic acid sequences, and, specifically, DNAmolecules and the amino acid encoded by the DNA molecules, which arecritical to male fertility. A promoter of the DNA is identified, as wellas its essential sequences. It also relates to use of such DNA moleculesto mediate fertility in plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a locus map of the male sterility gene BS92-7.

FIG. 2. is a gel of a Southern Blot analysis of EcoRI digested DNA froma Mu family segregating for male sterility and hybridized with a Mu1probe.

FIG. 3. is a Northern Blot analysis gel of total RNA from varioustissues hybridized with a PstI/BglII fragment from the BS92-7 clone.

FIG. 4 shows the nucleotide and protein sequences of the cDNA of BS92-7(The cDNA is SEQ ID NO: 1, the protein is SEQ ID NO: 2).

FIG. 5 is the genomic BS92-7 sequence (the nucleotide sequence is alsoreferred to as SEQ ID NO 3).

FIG. 6 is a comparisons of the genomic BS92-7 sequence with the cDNA(SEQ ID NO:3 and SEQ ID NO: 1); Part 1 is bases 301 to 450 of SEQ ID NO:3 and bases 1 to 117 of SEQ ID NO: 1. Part 2 is bases 501 to 750 of SEQID NO: 3 and bases 118 to 290 of SEQ ID NO: 1. Part 3 is bases 851 to1050 of SEQ ID NO: 3 and bases 291 to 487 of SEQ ID NO: 1. Part 4 isbases 1151 to 1350 of SEQ ID NO: 3 and bases 488 to 648 of SEQ ID NO: 1.Part 5 is bases 1401 to 1650 of SEQ ID NO: 3 and bases 649 to 841 of SEQID NO: 1. Part 6 is bases 1701 to 2140 of SEQ ID NO: 3 and bases 842 to1197 of SEQ ID NO: 1.

FIG. 7. is a Northern analysis gel showing developmental gene expressionin microsporogenesis of the gene BS92-7.

FIG. 8 is the full length promoter of BS92-7 (SEQ ID NO: 5)

FIG. 9. is a bar graph showing luciferase activity after substitution byrestriction site linker scanning of select small (9-10 bp) regions ofthe BS92-7 essential promoter fragment.

FIG. 10 shows an essential region of the BS92-7 promoter (SEQ ID NO: 6).

FIG. 11 shows a diagram of a construct used to express the MS45 malefertility gene using the BS7 promoter, and an immunoblot analysis of theMS45 protein from maize plants transformed with the construct.

FIG. 12 is a comparison of BS92-7 sorghum tassel (DNA is SEQ ID NO: 7and protein is SEQ ID NO: 8) and BS92-7 maize cDNA (DNA is bases 29 to731 of SEQ ID NO: 1 and protein is residues 10 to 244 of SEQ ID NO: 2).

DISCLOSURE OF THE INVENTION

All references referred to are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated therein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting.

Genetic male sterility results from a mutation, suppression, or otherimpact to one of the genes critical to a specific step inmicrosporogenesis, the term applied to the entire process of pollenformulation. These genes can be collectively referred to as malefertility genes (or, alternatively, male sterility genes). There aremany steps in the overall pathway where gene function impacts fertility.This seems aptly supported by the frequency of genetic male sterility inmaize. New alleles of male sterility mutants are uncovered in materialsthat range from elite inbreds to unadapted populations. To date,published genetic male sterility research has been mostly descriptive.Some efforts have been made to establish the mechanism of sterility inmaize, but few have been satisfactory. This should not be surprisinggiven the number of genes that have been identified as being responsiblefor male sterility. One mechanism is unlikely to apply to all mutations.

At U.S. Pat. No. 5,478,369 there is described a method by which a malesterility gene was tagged on maize chromosome 9. Previously, the onlydescribed male sterility gene on chromosome 9 was MS2, which has neverbeen cloned and sequenced. See Albertsen, M. and Phillips, R. L.,“Developmental Cytology of 13 Genetic Male Sterile Loci in Maize”Canadian Journal of Genetics &Cytology 23:195-208 (January 1981). Theonly fertility gene cloned before that had been the Arabadopsis genedescribed at Aarts, et al., supra.

The BS92-7 gene described herein is located on maize chromosome 7 and iscritical to male fertility. The locus map is represented at FIG. 1. Itcan be used in the systems described above, and other systems impactingmale fertility.

The maize family cosegregating for sterility was named BS92-7 and wasfound to have an approximately 7.0 Kb EcoRI fragment that hybridizedwith a Mu1 probe. A genomic clone from the family was isolated whichcontained a Mu1 transposon. A probe made from DNA bordering thetransposon was found to hybridize to the same ˜7.0 Kb EcoRI fragment.This probe was used to isolate cDNA clones from a tassel cDNA library.The cDNA for BS92-7 is 1230 bp, and the Mu insertion occurred in exon 2of the gene. Expression patterns, as determined by Northern analysis,show tassel specificity with peak expression highest at about thequartet to quartet release stages of microsporogenesis.

Further, it will be evident to one skilled in the art that variations,mutations, derivations including fragments smaller than the entiresequence set forth may be used which retain the male sterilitycontrolling properties of the gene. One of ordinary skill in the art canreadily assess the variant or fragment by its introduction into plantshomozygous for a stable male sterile allele of BS92-7, followed byobservation of the plant's male tissue development.

The invention also includes those nucleotide sequences which selectivelyhybridize to BS92.7 nucleotide sequences under stringent conditions. Inreferring to a sequence that “selectively hybridizes” with BS92-7, theterm includes reference to hybridization, under stringent hybridizationconditions, of a nucleic acid sequence to the specified nucleic acidtarget sequence to a detectably greater degree (e.g., at least 2-foldover background) than its hybridization to non-target nucleic acid.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare target-sequence-dependent and will differ depending on the structureof the polynucleotide. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to a probe (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, probes of this type are in arange of about 1000 nucleotides in length to about 250 nucleotides inlength.

An extensive guide to the hybridization of nucleic acids is found inTijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). See also Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. Generally, stringent wash temperature conditions areselected to be about 5° C. to about 2° C. lower than the melting point(Tm) for the specific sequence at a defined ionic strength and pH. Themelting point, or denaturation, of DNA occurs over a narrow temperaturerange and represents the disruption of the double helix into itscomplementary single strands. The process is described by thetemperature of the midpoint of transition, Tm, which is also called themelting temperature. Formulas are available in the art for thedetermination of melting temperatures.

Preferred hybridization conditions for the nucleotide sequence of theinvention include hybridization at 42° C. in 50% (w/v) formamide, 6×SSC, 0.5% (w/v) SDS, 100(g/ml) salmon sperm DNA. Exemplary lowstringency washing conditions include hybridization at 42° C. in asolution of 2× SSC, 0.5% (w/v) SDS for 30 minutes and repeating.Exemplary moderate stringency conditions include a wash in 2×SSC, 0.5%(w/v) SDS at 50° C. for 30 minutes and repeating. Exemplary highstringency conditions include a wash in 2× SSC, 0.5% (w/v) SDS, at 65°C. for 30 minutes and repeating. Sequences that correspond to thepromoter of the present invention may be obtained using all the aboveconditions. For purposes of defining the invention, the high stringencyconditions are used.

Promoter regions can be readily identified by one skilled in the art.The putative start codon containing the ATG motif is identified andupstream from the start codon is the presumptive promoter. By “promoter”is intended a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particular codingsequence. A promoter can additionally comprise other recognitionsequences generally positioned upstream or 5′ to the TATA box, referredto as upstream promoter elements, which influence the transcriptioninitiation rate. It is recognized that having identified the nucleotidesequences for the promoter region disclosed herein, it is within thestate of the art to isolate and identify further regulatory elementsupstream of the TATA box from the particular promoter region identifiedherein. Thus the promoter region disclosed herein is generally furtherdefined by comprising upstream regulatory elements such as thoseresponsible for tissue and temporal expression of the coding sequence,enhancers and the like. In the same manner, the promoter elements whichenable expression in the desired tissue such as male tissue can beidentified, isolated, and used with other core promoters to confirm maletissue-preferred expression.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the heterologousnucleotide sequence. Less than the entire promoter region can beutilized and the ability to drive anther-preferred expression retained.However, it is recognized that expression levels of mRNA can bedecreased with deletions of portions of the promoter sequence. Thus, thepromoter can be modified to be a weak or strong promoter. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. Generally, atleast about 30 nucleotides of an isolated promoter sequence will be usedto drive expression of a nucleotide sequence. It is recognized that toincrease transcription levels, enhancers can be utilized in combinationwith the promoter regions of the invention. Enhancers are nucleotidesequences that act to increase the expression of a promoter region.Enhancers are known in the art and include the SV40 enhancer region, the³⁵S enhancer element, and the like.

Smaller fragments may yet contain the regulatory properties of thepromoter so identified and deletion analysis is one method ofidentifying essential regions. Deletion analysis can occur from both the5′ and 3′ ends of the regulatory region. Fragments can be obtained bysite-directed mutagenesis, mutagenesis using the polymerase chainreaction and the like. (See, Directed Mutagenesis: A Practical ApproachIRL Press (1991)). The 3′ deletions can delineate the essential regionand identify the 3′ end so that this region may then be operably linkedto a core promoter of choice. Once the essential region is identified,transcription of an exogenous gene may be controlled by the essentialregion plus a core promoter. The core promoter can be any one of knowncore promoters such as the Cauliflower Mosaic Virus ³⁵S or 19S promoter(U.S. Pat. No. 5,352,605), ubiquitin promoter (U.S. Pat. No. 5,510,474)the IN2 core promoter (U.S. Pat. No. 5,364,780) or a Figwort MosaicVirus promoter (Gruber, et al. “Vectors for Plant Transformation”Methods in Plant Molecular Biology and Biotechnology Glick et al. eds,CRC Press pp. 89-119 (1993)).

The regulatory region of BS92-7 has been identified as including theabout 270 base pair region upstream of the putative TATA box. (See FIG.8.) Further, using the procedures outlined above, it has been determinedthat an essential region of the promoter includes the −112 to −93 bpupstream of the TATA box.

Promoter sequences from other plants may be isolated according towell-known techniques based on their sequence homology to the promotersequence set forth herein. In these techniques, all or part of the knownpromoter sequence is used as a probe which selectively hybridizes toother sequences present in a population of cloned genomic DNA fragments(i.e. genomic libraries) from a chosen organism. Methods are readilyavailable in the art for the hybridization of nucleic acid sequences.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequences.To achieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingpromoter sequences from a chosen organism by the well-known process ofpolymerase chain reaction (PCR). This technique can be used to isolateadditional promoter sequences from a desired organism or as a diagnosticassay to determine the presence of the promoter sequence in an organism.Examples include hybridization screening of plated DNA libraries (eitherplaques or colonies; see e.g. Innis et al., eds., (1990) PCR Protocols AGuide to Methods and Applications, Academic Press).

Further, the promoter of the present invention can be linked withnucleotide sequences other than the BS92-7 gene to express otherheterologous nucleotide sequences. The nucleotide sequence for thepromoter of the invention, as well as fragments and variants thereof,can be provided in expression cassettes along with heterologousnucleotide sequences for expression in the plant of interest, moreparticularly in the male tissue of the plant. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the nucleotide sequence to be under the transcriptional regulation ofthe promoter. These expression cassettes are useful in the geneticmanipulation of any plant to achieve a desired phenotypic response.Examples of other nucleotide sequences which can be used with the BS92-7promoter as the exogenous gene of the expression vector includecomplementary nucleotidic units such as antisense molecules (callaseantisense RNA, barnase antisense RNA and chalcone synthase antisenseRNA, Ms45 antisense RNA), ribozymes and external guide sequences, anaptamer or single stranded nucleotide sequences. The exogenousnucleotide sequence can also encode auxins, rol B, cytotoxins, diptheriatoxin, DAM methylase, avidin, or may be selected from a prokaryoticregulatory system. By way of example, Mariani, et al., Nature; Vol. 347;pp. 737; (1990), have shown that expression in the tapetum of eitherAspergillus oryzae RNase-T1 or an RNase of Bacillus amyloliquefaciens,designated “barnase,” induced destruction of the tapetal cells,resulting in male infertility. Quaas, et al., Eur. J. Biochem. Vol. 173:pp. 617 (1988), describe the chemical synthesis of the RNase-T1, whilethe nucleotide sequence of the barnase gene is disclosed in Hartley, J.Molec. Biol.; Vol. 202: pp. 913 (1988). The rolB gene of Agrobacteriumrhizogenes codes for an enzyme that interferes with auxin metabolism bycatalyzing the release of free indoles from indoxy-β-glucosides.Estruch, et al., EMBO J. Vol. 11: pp. 3125 (1991) and Spena, et al.,Theor. Appl. Genet.; Vol. 84: pp. 520 (1992), have shown that theanther-specific expression of the rolB gene in tobacco resulted inplants having shriveled anthers in which pollen production was severelydecreased and demonstrated the rolB gene is another gene that is usefulfor the control of pollen production. Slightom, et al., J. Biol. Chem.Vol. 261: pp. 108 (1985), disclose the nucleotide sequence of the rolBgene. DNA molecules encoding the diphtheria toxin gene can be obtainedfrom the American Type Culture Collection (Rockville, Md.), ATCC No.39359 or ATCC No. 67011 and see Fabijanski, et al., E.P. Appl. No.90902754.2, “Molecular Methods of Hybrid Seed Production” for examplesand methods of use. The DAM methylase gene is used to cause sterility inthe methods discussed at U.S. Pat. Nos. 5,792,853, 5,689,049 andPCT/US95/15229 Cigan, A. M. and Albertsen, M. C., “Reversible NuclearGenetic System for Male Sterility in Transgenic Plants”. Also seediscussion of use of the avidin gene to cause sterility at U.S. Pat. No.5,962,769.

The invention includes vectors with the BS92-7 gene. A vector isprepared comprising the BS92-7 gene, a promoter that will driveexpression of the gene in the plant and a terminator region. As noted,the promoter in the construct may be the native promoter or asubstituted promoter which will provide expression in the plant. Thechoice of promoter will depend upon the use intended of the gene. Thepromoter in the construct may be an inducible promoter, so thatexpression of the sense or antisense molecule in the construct can becontrolled by exposure to the inducer.

Other components of the vector may be included, also depending uponintended use of the gene. Examples include selectable markers, targetingor regulatory sequences, stabilizing or leader sequences, etc. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., “Vectors for Plant Transformation” inMethod in Plant Molecular Biology and Biotechnology, Glick et al eds;CRC Press pp. 89-119 (1993). The selection of an appropriate expressionvector will depend upon the host and the method of introducing theexpression vector into the host. The expression cassette can alsoinclude at the 3′ terminus of the heterologous nucleotide sequence ofinterest, and a transcriptional and translational termination regionfunctional in plants. The termination region can be native with thepromoter nucleotide sequence of the present invention, can be nativewith the DNA sequence of interest, or can be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al. Mol. Gen. Genet.262:141-144 (1991); Proudfoot Cell 64:671-674 (1991); Sanfacon et al.Genes Dev. 5:141-149 (1991); Mogen et al. Plant Cell 2:1261-1272 (1990);Munroe et al. Gene 91:151-158 (1990); Ballas et al. Nucleic Acids Res.17:7891-7903 (1989); Joshi et al. Nucleic Acid Res. 15:9627-9639 (1987).

The expression cassettes can additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picomavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region),Elroy-Stein et al. Proc. Nat. Acad. Sci. USA 86:6126-6130 (1989);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allisonet al.; MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20(1986); human immunoglobulin heavy-chain binding protein (BiP), Macejaket al. Nature 353:90-94 (1991); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. Nature325:622-625 (1987); Tobacco mosaic virus leader (TMV), Gallie et al.(1989) Molecular Biology of RNA, pages 237-256; and maize chloroticmottle virus leader (MCMV) Lommel et al. Virology 81:382-385 (1991). Seealso Della-Cioppa et al. Plant Physiology 84:965-968 (1987). Thecassette can also contain sequences that enhance translation and/or mRNAstability such as introns.

In those instances where it is desirable to have the expressed productof the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast, or to the endoplasmicreticulum, or secreted at the cell's surface or extracellularly, theexpression cassette can further comprise a coding sequence for a transitpeptide. Such transit peptides are well known in the art and include,but are not limited to, the transit peptide for the acyl carrierprotein, the small subunit of RUBISCO, plant EPSP synthase, and thelike. One skilled in the art will readily appreciate the many optionsavailable in expressing a product to a particular organelle. Forexample, the barley alpha amylase sequence is often used to directexpression to the endoplasmic reticulum (Rogers, J. Biol. Chem.260:3731-3738 (1985)). Use of transit peptides is well known (e.g., seeU.S. Pat. Nos. 5,717,084; 5,728,925).

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions, such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the promoter. Ingeneral, the vectors should be functional in plant cells. At times, itmay be preferable to have vectors that are functional in E. Coli (e.g.,production of protein for raising antibodies, DNA sequence analysis,construction of inserts, obtaining quantities of nucleic acids). Vectorsand procedures for cloning and expression in E. Coli are discussed inSambrook et al. (supra).

The transformation vector comprising the promoter sequence of thepresent invention operably linked to a heterologous nucleotide sequencein an expression cassette can also contain at least one additionalnucleotide sequence for a gene to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

Reporter genes can be included in the transformation vectors. Examplesof suitable reporter genes known in the art can be found in, forexample, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuescan be included in the transformation vectors. These can include genesthat confer antibiotic resistance or resistance to herbicides. Examplesof suitable selectable marker genes include, but are not limited to,genes encoding resistance to chloramphenicol, Herrera Estrella et al.(1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983)Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820;hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian etal. (1995) Plant Science 108:219-227; streptomycin, Jones et al (1987)Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard et al.(1996) Transgenic Res. 5:131-137; bleomycin, Hille et al. (1990) PlantMol. Biol. 7:171-176; sulfonamide, Guerineau et al. (1990) Plant Mol.Biol. 15:127-136; bromoxynil, Stalker et al. (1988) Science 242:419-423;glyphosate, Shaw et al. (1986) Science 233:478-481; phosphinothricin,DeBlock et al. (1987) EMBO J. 6:2513-2518.

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription or transcript andtranslation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for efficienttransformation/transfection may be employed.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki et al, “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biotechnology supra; Klein et al,(1992) Bio/Technology 10:268; and Weising et al., (1988) Ann. Rev.Genet. 22: 421-477. For example, the DNA construct may be introducedinto the genomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery, Klein et al., (1987) Nature 327:70-73; electroporation, Fromm et al., (1985) Proc. Natl. Acad. Sci. 82:5824; polyethylene glycol (PEG) precipitation, Paszkowski et al., (1984)EMBO J. 3: 2717-2722; direct gene transfer WO 85/01856 and EP No. 0 275069; in vitro protoplast transformation U.S. Pat. No. 4,684,611; andmicroinjection of plant cell protoplasts or embryogenic callus.Crossway, (1985) Mol. Gen. Genetics 202:179-185. Co-cultivation of planttissue with Agrobacterium tumefaciens is another option, where the DNAconstructs are placed into a binary vector system. See e.g., U.S. Pat.No. 5,591,616; Ishida et al., (1996) Nature Biotechnologv 14:745-750.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct into the plant cell DNA when thecell is infected by the bacteria. See, for example Horsch et al., (1984)Science 233: 496-498, and Fraley et al., (1983) Proc. Natl. Acad. Sci.80: 4803.

Standard methods for transformation of canola are described at Moloneyet al. “High Efficiency Transformation of Brassica napus usingAgrobacterium Vectors” Plant Cell Reports 8:238-242 (1989). Corntransformation is described by Fromm et al, Bio/Technology 8:833 (1990)and Gordon-Kamm et al, supra. Agrobacterium is primarily used in dicots,but certain monocots such as maize can be transformed by Agrobacterium.See supra and U.S. Pat. No. 5,550,318. Rice transformation is describedby Hiei et al., (1992) The Plant Journal 6(2): 271-282 (1994, Christouet al, Trends in Biotechnology 10:239 and Lee et al, Proc. Nat'l Acad.Sci. USA 88:6389 (1991). Wheat can be transformed by techniques similarto those used for transforming corn or nce. Sorghum transformation isdescribed at Casas et al, supra and sorghum by Wan et al, (1994) PlantPhysicol. 104:37. Soybean transformation is described in a number ofpublications, including U.S. Pat. No. 5,015,580.

Further detailed description is provided below by way of instruction andillustration and is not intended to limit the scope of the invention.

EXAMPLE 1 Identification and Cosegregation of BS92-7

Families of plants from a mutator (Mu) population were identified thatsegregated for a male sterile phenotype, with none or only a fewextruded abnormal anthers, none of which had pollen present. Malesterility is expected to result from those instances where a Mu elementhas randomly integrated into a gene responsible for some step inmicrosporogenesis, disrupting its expression. Plants from a segregatingF₂ family, designated BS92-7, were grown and classified for malefertility/sterility based on the above criteria. Leaf samples were takenand subsequent DNA isolated on approximately 20 plants per phenotypicclassification

Southern analysis was performed to confirm association of Mu withsterility. Southern analysis is a well known technique to those skilledin the art. This common procedure involves isolating the plant DNA,cutting with restriction endonucleases, fractionating the cut DNA bymolecular weight on an agarose gel, and transferring to nylon membranesto fix the separated DNA. These membranes were subsequently hybridizedwith the Mu-probe fragment that was radioactively labeled withα³²P-dCTP, and washed in an SDS solution. Southern, E., (1975)“Detection of Specific Sequences Among DNA Fragments by GelElectrophoresis,” J. Mol. Biol. 98:503-317. Plants from a segregating F₂BS92-7 family were grown and classified for male fertility/sterility.Leaf sampling and subsequent DNA isolation was accomplished onapproximately 20 plants per phenotypic classification. DNA (˜7 ug) from5 fertile and 12 sterile plants was digested with EcoRI and subjected toelectrophoresis through a 0.75% agarose gel. The digested DNA wastransferred to nylon membrane via Southern transfer. The membrane washybridized with an internal fragment from the Mu1 transposon.Autoradiography of the membrane revealed cosegregation of a 7 Kb EcoRIfragment with the sterility phenotype as shown at FIG. 2. This EcoRIband segregated in the fertile plants suggesting a segregatinghomozygous-heterozygous wild type condition for the allele.

EXAMPLE 2 Library Construction and Screening

The process of cDNA library screenings is commonly known among thoseskilled in the art and is described at Sambrook, J., Fritsch, E. F.,Maniatis T., et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Lab Press, Plainview, N.Y.Libraries were created as follows.

DNA from a sterile plant was digested with EcoRI and run on apreparative gel. DNA with a molecular weight between 6.0 and 8.0 kb wasexcised from the gel, electroeluted and ethanol precipitated. This DNAwas ligated into the Lambda Zap vector (Stratagene™) using themanufacturer's protocol. The ligated DNA was packaged into phageparticles using Gigapack Gold (Stratagene™). Approximately 500,000 PFUwere plated and lifted onto nitrocellulose membranes. Membranes werehybridized with the Mu1 probe. A pure clone was obtained after threerounds of screening. The insert was excised from the phage as a plasmidand designated BS927-8.1. A border fragment from this clone was isolatedand used to reprobe the original EcoRI cosegregation blot. The 7.0 kbEcoRI fragment is homozygous in all the sterile plants, which confirmsthat the correct Mu fragment was isolated. Eight of the fertile plantsare heterozygous for the 7.0 kb EcoRI band and a 6.2 Kb EcoRI band. Twoof the fertile plants are homozygous for the 6.2 kb EcoRI band,presumably the wild type allele.

EXAMPLE 3 Expression Analysis and cDNA Isolation

Northern analysis can be used to detect expression of genes at variousstages of microsporogenesis. Northern analysis is a commonly usedtechnique known to those skilled in the art and is similar to Southernanalysis except that mRNA instead of DNA is isolated and placed on thegel. The mRNA is then hybridzed with the labeled probe. Potter, E., etal., (1981) Proc. Nat. Acad. Sci. USA 78:6662-6666, Lechelt, et al.(1989) Mol. Gen. Genet. 219:225-234. A PstI/BgIII fragment fromBS927-8.1 was used to probe a northern blot containing kernel, immatureear, seedling and tassel RNA. A signal was seen only in tassel RNA atapproximately the quartet stage of microsporogenesis as reflected atFIG. 3. The transcript is about 1.4 kb in length. The same probe wasalso used to screen a cDNA library constructed from mRNA isolated frommeiotic to late uninucleate staged anthers. Two clones, designatedBS927-4.1 and BS927-9.1, were isolated from the library.

EXAMPLE 4 Sequence Analysis

BS927-8.1 genomic clone and the two cDNA clones, BS927-4.1 andBS927-9.1, were sequenced. Sequences methods are well known in the artand this sequencing was accomplished by Loftstrand Labs Limited usingthe methods discussed at Sanger, F., Nicklen, S., Coulson, A. R. (1977)“DNA Sequences with chain terminating inhibitors” Proc. Natl. Acad. Sci.USA 74:5463-5467. The two cDNA clones differ at the 5′ end of themolecule. BS927-4.1 contains a TCTC repeat, whereas the BS927-9.1 doesnot. When these sequences are compared to the genomic clone, the TCTCrepeat is not present and probably represents a cloning artifact in theBS927-4.1 cDNA. The cDNA/genomic comparison reveals six exons and fiveintrons are present in the genomic clone. The cDNA sequence is set forthin FIG. 4 and the genomic shown in FIG. 5. A comparison of the genomicand cDNA is provided in FIG. 6 and demonstrated a 95.95% identity. TheMu1 insertion occurs in exon2. There is a putative Met start codon atposition 320 in the genomic clone. Since both cDNAs lack this Met codon,they did not represent full length genes. Subsequent cDNA screening withBS927-4.1 allowed for the isolation of clone BS927-11.1. This clone wasonly sequenced at the 5′ end to determine its start point. It wasdetermined that BS927-11.1 lacks 2 bases of the Met codon (ATG) and isthe longest cDNA isolated. Further expression studies were done usingthe BS927-4.1 cDNA probe against a Northern containing mRNA at discretestages of microsporogenesis. Signal is detected from meiosis II/quartetto mid-uninucleate, with maximal signal being at quartet to quartetrelease as shown at FIG. 7.

EXAMPLE 5 Identification of Promoter and its Essential Regions

Comparison of the BS927-8.1 genomic clone with the cDNA clonesBS927-11.1, BS927-4.1 and BS927-9.1 allowed identification of intronsand exons in BS927. This in turn permitted identification of ORFs, oneof which extended through most of the cDNA sequence, and was most likelythe protein coding sequence of the BS927 gene. Testing for codonpreference and non-randomness in the third position of each codonconfirmed that this was the likely protein-coding ORF. At the amino acidlevel, the protein that would be encoded has 52% similarity (42%identity) with the maize gene A1, which encodes dihydroflavanolreductase and is required for synthesis of anthocyanins andphlobaphenes.

Regulatory regions of anther genes, such as promoters, may be identifiedin genomic subclones using functional analysis, usually verified by theobservation of reporter gene expression in anther tissue and a lowerlevel or absence of reporter gene expression in non-anther tissue. Thepossibility of the regulatory regions residing “upstream” or 5′ ward ofthe transcriptional start site can be tested by subcloning a DNAfragment that contains the upstream region into expression vectors fortransient expression experiments. It is expected that smaller subgenomicfragments may contain the regions essential for male-tissue preferredexpression. For example, the essential regions of the CaMV 19S and ³⁵Spromoters have been identified in relatively small fragments derivedfrom larger genomic pieces as described in U.S. Pat. No. 5,352,605.

The selection of an appropriate expression vector with which to test forfunctional expression will depend upon the host and the method ofintroducing the expression vector into the host and such methods arewell known to one skilled in the art. For eukaryotes, the regions in thevector include regions that control initiation of transcription andcontrol processing. These regions are operably linked to a reporter genesuch as UidA, encoding—glucuronidase (GUS), or luciferase. Generaldescriptions and examples of plant expression vectors and reporter genescan be found in Gruber, et al., supra. GUS expression vectors and GUSgene cassettes are commercially available from Clonetech, Palo Alto,Calif., while luciferase expression vectors and luciferase genecassettes are available from Promega Corporation, Madison, Wis. Tiplasmids and other Agrobacterium vectors are described in Ishida, Y., etal., (1996) Nature Biotechnology; Vol. 14; pp. 745-750; and in U.S. Pat.No. 5,591,616.

Expression vectors containing putative regulatory regions located ingenomic fragments can be introduced into intact tissues such as stagedanthers, embryos or into callus. Methods of DNA delivery includemicroprojectile bombardment, DNA injection, electroporation andAgrobacterium-mediated gene transfer (see Gruber, et al., supra, U.S.Pat. No. 5,591,616, and Ishida, Y., et al., supra). General methods ofculturing plant tissues are found in Gruber, et al., supra.

For the transient assay system, staged, isolated anthers are immediatelyplaced onto tassel culture medium (Pareddy, D. R. and J. F. Petelino,(1989) Crop Sci. J.; Vol. 29; pp. 1564-1566;) solidified with 0.5%Phytagel (Sigma, St. Louis) or other solidifying media. The expressionvector DNA is introduced within 5 hours preferably bymicroprojectile-mediated delivery with 1.2 μm particles at 1000-1100Psi. After DNA delivery, the anthers are incubated at 26° C. upon thesame tassel culture medium for 17 hours and analyzed by preparing awhole tissue homogenate and assaying for GUS or for lucifierase activity(see Gruber, et al., supra).

Upstream of the likely translational start codon of BS927, only 319 bpof BS927 DNA were present in the genomic clone BS927-8.1. Translationalfusions via an engineered NcoI site were generated with reporter genesencoding luciferase and β-glucuronidase to test whether this fragment ofDNA had promoter activity in transient expression assays of bombardedplant tissues. Activity was demonstrated in anthers and not incoleoptiles, roots and calli, suggesting anther-preferred oranther-specific promoter activity.

A reasonable TATA box was observed by inspection upstream of thetranslational start codon. The genomic clone BS927-8.1 thus includesonly about 266 bp upstream of the putative TATA box. For typical plantgenes, the start of transcription is 26-36 bp downstream of the TATAbox, which would give the BS927 mRNA a 5′-nontranslated leader of onlyabout 17-27 nucleotides (nt). The total BS927 subgenomic fragment ofonly 319 bp, including nontranslated leader, start of transcription,TATA box and sequences upstream of the TATA box, was thus shown to besufficient for promoter activity. See FIG. 8, which is SEQ. ID NO.5. Itwill be appreciated by those skilled in the art that promoter fusionswith genes, open reading frames, RNA-encoding sequences and the like maybe either at or close to the native start of transcription or at thestart of translation or downstream of the start codon. The putative TATAbox (TTTATAA) is underlined. Thus, the present invention encompasses aDNA molecule having a nucleotide sequence of SEQ ID NO. 5 (or those withsequence identity or which hybridize thereto) and having the function ofa male tissue-preferred regulatory region.

Deletion analysis can occur from both the 5′ and 3′ ends of theregulatory region: fragments can be obtained by site-directedmutagenesis, mutagenesis using the polymerase chain reaction, and thelike (Directed Mutagenesis: A Practical Approach; IRL Press; (1991)).The 3′ end of the male tissue-preferred regulatory region can bedelineated by proximity to the putative TATA box or by 3′ deletions ifnecessary. The essential region may then be operably linked to a corepromoter of choice via cloning restriction sites introduced into theregion immediately upstream of the TATA box or as defined by 3′ deletionanalysis. Once the essential region is identified, transcription of anexogenous gene may be controlled by the male tissue-preferred region ofBS92-7 plus a core promoter. The core promoter can be any one of knowncore promoters such as a Cauliflower Mosaic Virus ³⁵S or 19S promoter(U.S. Pat. No. 5,352,605), ubiquitin (U.S. Pat. No. 5,510,474), the IN2core promoter (U.S. Pat. No. 5,364,780), or a Figwort Mosaic Viruspromoter (Gruber, et al., supra). Preferably, the promoter is the corepromoter of a male tissue-preferred gene or the CaMV 35S core promoter.More preferably, the promoter is from a male tissue-preferred gene andin particular, the BS92-7 core promoter.

Further mutational analysis, for example by linker scanning, a methodwell-known to the art, can identify small segments containing sequencesrequired for anther-preferred expression. These mutations may introducemodifications of functionality such as in the levels of expression, inthe timing of expression or in the tissue of expression. Mutations mayalso be silent and have no observable effect.

The foregoing procedures were used to identify essential regions of theBS92-7 promoter. After linking the promoter with the luciferase markergene, mutational analyses were performed. The 319 bppromoter/nontranslated leader region of the genomic clone BS7 comprises266 bp upstream of the putative TATA box. Deletion of the upstream-mostone half (approximately) of this upstream region to an MluI site reducedtransient promoter activity about tenfold in anthers, although activitywas not eliminated. Introduction of a BglII site at −15 to −10 relativeto the putative TATA box did not greatly affect activity, but thecombination of this cloning site modification with the deletioneliminated activity.

Linker scanning was initiated from −261 through −16 relative to theputative TATA box, mostly in 10 bp increments, as represented in FIG. 9.The bar graph shows each 7-10 bp substituted segment on the x-axis. They-axis shows the normalized luciferase activity as a percent of wildtype promoter activity. The linker scanning constructs and wild typecontrol included the BglII cloning site. The minimal promoter is furtherrepresented in FIG. 10. The TATA box is identified by underlining. Asingle critical region from −112 through −93 relative to the putativeTATA box was observed, with additional regions having a significantimpact located at −161 to −152; −141 to −132; and −92 to −83 relative tothe putative TATA box. The region from about −102 to −93 relative to theputative TATA box includes two overlapping copies of a sequence, the Pmotif, implicated in the function of other anther promoter upstreamregions as well as promoters activated by the maize P gene product. Themyb-homologous P gene controls phlobaphene pigmentation in maize floralorgans by directly activating a flavonoid biosynthetic gene subset.(Grotewald, E., B. J. Drummond, B. Bowen, and T. Peterson (1994) Cell76:543-553.)

EXAMPLE 6 BS92-7 Promoter Used to Drive MS45 Gene

The MS45 gene is a male fertility gene in maize and mutations in thegene result in breakdown of microsporogenesis during vacuolation of themicrospores rendering the plants male sterile. When the cloned maizeMS45 gene is introduced into such mutated male sterile plants, the genecan complement the mutation and confer male fertility. For a completediscussion of the MS45 gene and its promoter, see U.S. Pat. Nos.5,478,369 and 6,037,523, incorporated herein by reference. PHP6641 is apUC8 plasmid containing the MS45 gene promoter and coding regionincluding introns, nucleotides 1-3335 cloned as NcoI DNA fragmentupstream of the ³⁵S::PAT selectable marker gene as described above. Sitedirected mutagenesis (as per Su T. S. and El-Gewely M. R. supra, wasused to introduce a NcoI restriction enzyme recognition site at thetranslation start codon of the MS45 gene (nucleotide 1389). A 4.7 kpHindIII-EcoRI DNA fragment containing the mutagenized version ofMS45-35S::PAT was cloned in plasmid pSB11 (pSB31 from Ishida et alsupra.) lacking the EcoRI DNA fragment insert carrying the ³⁵S GUS and35SBAR genes resulting in PHP10890. To produce PHP12025, the BS92.7promoter replaced the MS45 promoter. This was then introduced into aconstruct, shown in FIG. 11. This construct was introduced byAgrobacterium-mediated transformation as described above into a mutantmaize plant which was male sterile as a result of a mutation of the MS45gene. All 22 events generated from the transformation were restored to amale-fertile phenotype. Immunoblot analysis of the MS45 protein from theplants is also shown in FIG. 11. MS45 protein from an inbred expressingwild-type MS45 gene served as the control. Protein extracts wereisolated from anthers staged at tetrad release to early vacuolate stagesof microspore development. The MS45 protein was expressed to nearlywild-type levels when transcribed from the maize BS92.7 promoter. Thusthe BS92.7 promoter was able to drive appropriate expression of a generequired to restore fertility to a male-sterile mutant line. Table 1summarizes the results below. TABLE 1 % Male- % Male- Number ofConstruct Description Fertile Sterile events PHP10890 MS45::MS45 100% 11PHP12205 BS92.7::MS45 100% 22

EXAMPLE 7 Impacting Male Fertility Using the BS7 Promoter

In addition, the BS92.7 promoter was able to drive expression of acytotoxic gene to confer male sterility on a male-fertile genotype. Theconstructs to produce male sterile plants were developed as follows. TheE. coli DNA adenine methylase gene (DAM gene) as described at U.S. Pat.No. 5,792,853, was used. MS45:DAM-35SPAT(PHP12634) The DAM gene wasmodified by site-directed mutagenesis (Su and El-Gewley, (1988) Gene69:81-89) and a SmaI site introduced at nucleotide 186, nine nucleotides5′ to the initiating codon ATG of the DAM gene. The NcoI site of a 1.4kb HindIII-NcoI fragment containing the maize MS45 promoter found onplasmid PHP6054 was filled-in with dNTPs using T4 DNA polymerase andligated to the SmaI site contained at the 5′ end of the DAM gene.Transcription of this gene was terminated by the addition of the 3′sequences from the potato proteinase inhibitor II gene (PinII)(nucleotides 2-310; An et al., (1989) Plant Cell 1:115-122).

To construct the maize transformation vector PHP12634(MS45::DAM-35SPAT), the 2.5 kb chimeric gene containing the MS45promoter, the DAM gene and PinII 3′ non-translated region was cloned asa HindIII-NcoI fragment upstream of the ³⁵S::PAT gene in the vectorpSB11 (pSB31 from Ishida et al., supra, lacking the EcoRI fragmentinsert carrying the 35SGUS and 35SBAR genes). The PAT gene encodes theenzyme phosphinothricin acetyl-transferase (PAT) from Streptomycesviridochomagenes (nucleotides 6 to 557). See, EP 0 275 957 A; Genbankaccession number A02774. It was placed under the transcriptionalregulation of the cauliflower mosaic virus (CAMV) ³⁵S promoter andterminator regions (nucleotides 6906 to 7439, and 7439-7632). See U.S.Pat. No. 5,352,605 and Franck et al., (1980) Cell 21:285-294. The³⁵S::PAT component was contained on an NcoI-KpnI fragment as describedin the expression cassette pDH51 (Pietrzak et al., (1986) Nucleic AcidsRes. 14:5857-5868).

PHP12635 (BS7:DAM/35S:PAT) was constructed by replacing the MS45promoter in PHP12634 with the BS7 promoter.

In the following experiment the BS92.7 promoter was used to direct thetranscription of the sterility gene, Dam-methylase (DAM). A constructusing the MS45 promoter for transcription of DAM was also transformedinto wild-type maize. Table 2 below reflects that 100% of the eventscontaining BS92.7::DAM were male sterile, compared to the MS45::DAMconstruct in which only 25% of the events were male sterile. TABLE 2 %Male- % Male- Number of Construct Description Fertile Sterile eventsPHP12634 MS45::DAM 75%  25% 24 PHP12635 BS92.7::DAM 0 100% 10

EXAMPLE 8 Allelism

The BS927-8.1 clone was mapped in an ECB RFLP population using EcoRI asthe enzyme. The clone maps on the long arm of chromosome 7 between themolecular markers bnl15.40 and umc110a. The male sterile mutant, ms7, isthe only known male sterile that maps to chromosome 7. Allelism crosseswere initiated with the BS92-7 mutant and the ms7 stock. Progeny fromthis cross segregated for male sterility indicating that the same geneis responsible for the mutant phenotypes in both the BS92-7 and ms7families.

EXAMPLE 9 ms7 Isolation

Clone ms7-5.1 was purified and sequenced using internal primers alreadyconstructed for the BS92-7 sequencing work. There is one extra Serresidue in the ms7 deduced protein as compared to the BS92-7. The majordifference between the two is a 33 bp deletion in the promoter region ofms7. Transient assays of the ms7 promoter fragment show that it isactive in anthers.

As noted above, the BS92-7 nucleotide sequence is allelic to the knownmaize male sterile mutation ms7. When BS92-7 is mutated, male sterilitywill result. It may be introduced into plants to impact male sterility.

EXAMPLE 10 BS92-7 Sorghum Tassel RT-PCR and BS92-7 Maize cDNA Comparison

A homologue of BS92-7 was identified in sorghum. The sorghum-BS92-7 cDNAwas isolated by using the maize BS92-7 gene primers in a polymerasechain reaction with sorghum flower cDNA as the template. The resultantcDNA fragment was sequenced by methods described supra and then comparedto the BS92-7 cDNA from maize. Nucleotide sequence comparisons are setforth in FIG. 12, which shows 89.1% identity between the nucleotidesequences and 94% identity between the predicted protein sequences.

As is evident from the above, the BS92-7 gene is critical to malefertility in plants.

Thus it can be seen that the invention achieves at least all of itsobjectives.

1-34. (canceled)
 35. An isolated nucleic acid sequence comprising thepromoter region of the gene BS92-7.
 36. An isolated nucleic acidsequence comprising those nucleotide sequences which hybridize to SEQ IDNO: 5 under conditions of high stringency with a wash in 0.1×SSC, 0.1%(w/v) SDS, at 65° C. and which is essential for male tissue-preferredexpression of the BS92-7 gene.
 37. A male tissue-preferred regulatoryregion comprising a fragment of SEQ ID NO: 5 or SEQ ID NO: 6, whereinthe sequence is essential for male tissue-preferred regulation of asequence operably linked to said region.
 38. An isolated maletissue-preferred regulatory region comprising those nucleotide sequenceswhich hybridize to SEQ ID NO: 6 under conditions of high stringency witha wash in 0.1×SSC, 0.1% (w/v) SDS, at 65° C. and which is essential formale tissue-preferred expression of the BS92-7 gene.
 39. An isolatednucleic acid that is a male tissue-preferred regulatory regioncomprising those nucleotide sequences which hybridize to SEQ ID. NO: 6under conditions of high stringency, with a wash in 0.1× SSC, 0.1% (w/v)SDS, at 65° C. for 30 minutes wherein the regulatory region is essentialfor initiating transcription of SEQ ID NO: 1, 3 or 7 and which isessential for male tissue-preferred expression of SEQ ID NO: 1, 3 or 7.40-45. (canceled)
 46. An expression vector comprising a promoter that isoperably linked with the male tissue-preferred regulatory region ofclaim
 39. 47. The expression vector of claim 46 further comprising anexogenous sequence, wherein the exogenous sequence is operably linked tothe promoter.
 48. The expression vector of claim 46, wherein thepromoter is selected from the group consisting of the promoters ofCaMV35S, SGB6, BS92-7, MS45 or
 5126. 49. The expression vector of claim47, wherein the product of the exogenous gene disrupts the function ofmale tissue.
 50. Plant cells comprising the vector of claim
 46. 51. Amethod of mediating male fertility in a plant, comprising introducinginto a plant the expression vector of claim 47, wherein the exogenoussequence inhibits or restores male fertility of the plant and theregulatory region in conjunction with the promoter controls expressionof the exogenous sequence.
 52. The method of claim 51, wherein theexogenous sequence inhibits function of male tissue of the plant,causing the plant to be male sterile.
 53. The method of claim 52,wherein a regulatory element in conjunction with the promoter isinducible.
 54. The method of claim 53, wherein the plant isconstitutively sterile when the promoter and regulatory region are notinduced and is fertile when the promoter and regulatory region areinduced.
 55. The method of claim 51, further comprisingcross-fertilizing the male sterile plant with a second plant, the secondplant comprising a second exogenous sequence, the product of the secondsequence preventing disruption of the male tissue by the first exogenoussequence, producing a male fertile hybrid plant.
 56. A method ofproducing hybrid seeds comprising: (a) producing a first parent plantcomprising nucleotide sequence of claim 39 operably linked with anexogenous sequence impacting male fertility of the plant such that theplant is male sterile; (b) producing a second parent plant which is malefertile; and (c) cross-fertilizing the first parent plant and the secondparent plant to produce hybrid seeds.
 57. The method of claim 56,wherein the sequence impacting male fertility is dominant and whereinthe method further comprises growing the hybrid seed to produce a thirdmale sterile parent plant; producing a fourth parent plant comprisingone or more genes controlling a desired gene trait and cross-fertilizingthe third and fourth parent plants to produce second hybrid seed.